The pools of size-selected inserts were then utilized to construct libraries in the D-bio vector. Open in a separate window Figure 2 Size selection of brain cDNA library inserts. binding properties of the real ligand, and will hence interfere with the selection end result. Here we describe a novel lambda vector for display of exogenous peptides at the C-terminus of the capsid D protein. In this vector, translation of fusion peptides in the correct reading frame allows efficient 5-Iodo-A-85380 2HCl biotinylation of the chimeric phage during amplification. By using this vector system we constructed three libraries from human hepatoma cells, mouse hepatocytic MMH cells and from human brain. Clones containing open reading frames (ORFs) were rapidly selected by streptavidin affinity chromatography, leading to biological repertoires highly enriched in natural polypeptides. We compared the selection end result of two 5-Iodo-A-85380 2HCl impartial experiments performed using an anti-GAP-43 monoclonal antibody around the human brain cDNA library before and after ORF enrichment. A significant increase in the efficiency of identification 5-Iodo-A-85380 2HCl of natural target peptides with very little background of false-positive clones was observed in the latter case. INTRODUCTION Building a comprehensive map of proteinCprotein or 5-Iodo-A-85380 2HCl nucleic acidCprotein interactions is a major target in post-genomic research, and in the last few years a number of different strategies have been adopted for the quick identification of potential conversation partners (1C3). Affinity selectable biological repertoires have been successfully exploited to identify ligands for several ligates (4,5). Filamentous phage M13/fd has been proven to be the vector of choice to generate small peptide libraries or display specialized repertoires where variability is usually confined to a few amino acids in the context of a fixed scaffold (i.e. antibody libraries). In contrast, M13 display of cDNA libraries has met with only limited success, presumably due to some peculiar biological features of this phage, such as the requirement for the fusion products to be secreted prior to phage assembly (6). This requirement may expose a bias during phage production because of 5-Iodo-A-85380 2HCl inefficient recombinant protein translocation (7), which in turn would lead to under representation, or even the absence, of many polypeptides in the library. Thus, the M13 phage is not the ideal presentation vehicle for complex repertoires from natural sources, such as cDNA libraries. As an alternative to filamentous phage Rabbit polyclonal to ZNF404 a few laboratories, including our own, have chosen lytic phage as display vectors for exogenous proteins. With these vectors, encapsidation of the fusion protein is an intracellular event, thus making assembly of chimeric phage a less demanding process. Both T7 and lambda have been reported as suitable systems to expose large polypeptides of different nature (8C15). We previously explained the construction and affinity selection of cDNA expression libraries from viruses and bacteria, as well as complex repertoires from mammalian tissues or whole organisms, which were displayed around the lambda surface as fusion to the C-terminus of the D protein (16,17). Even though these libraries can be successfully surveyed with mono or polyclonal antibodies and even with proteins as baits (15), a large number of clones representing peptide mimics that specifically bind to the selector molecules were often isolated, and in some cases overcame selection of the natural ligand (17). These clones are generated by fusions of gene fragments to the D protein in a different frame from the correct one, but eventually give rise to short additions to the C-terminus of the D protein. To solve this problem, we have generated a new lambda display vector where the sequence for any 13 amino acid long peptide representing the target for the biotinylating BirA enzyme is usually engineered downstream of the cloning site. In this way, only cDNA fragments in-frame with both D and tag sequences will lead to the corresponding chimeric phage being biotinylated in strains made up of the BirA activity. We generated three libraries from mouse hepatic cells, human hepatoma and human brain, and demonstrated efficient separation of open reading frame (ORF)-encoding phage from non-ORF clones by affinity selection on streptavidin (SA) beads. ORF-enriched libraries were shown to perform much better than the untagged libraries in selection experiments. MATERIALS AND METHODS D-bio vector construction The DNA sequence coding for the 13 amino acid peptide LNDIFEAQKIEWH was inserted at the 3 of the lambda D gene in plasmid 171 (17), by ligation of the following annealed oligos: OL197, 5-CTAGTTTTTAATTGCGGCCGCGTGGTTCAGGCCTGAACGACATCTCGAAGCTCAGAAAATCGAATGGCACTAATCGGCCGC-3; OL198, 5-GGCCGATTAGTGCCATTCGATTTTCTGAGCTTCGAAGATGTCGTTAGGCCTGAACCACGCGGCCGCAATTAAAAA-3 to the using the Ready to Go Lambda Packaging Kit (Amersham Pharmacia Biotech) according to the provided instructions. Finally, a PCR fragment made up of the -lactamase gene and ColE1 ori was cloned into the ID-bio vector, to generate the D-bio vector utilized for the construction of the cDNA libraries. The PCR fragment was obtained by amplification of 171 plasmid with OL8 (5-TGCTTAATTAATGCAGCCCGGGCTCAAATTAAGCAGAAGGCCATCCT-3) made up of the packaged (Amersham Pharmacia Biotech). The packaging mixture was then utilized for infecting BB4 cells (OD600 = 2.00), plated into 100 square (23 23 cm) plates. Phage elution was achieved by scraping the agarose from your plates and shaking.